This invention relates to a novel method for producing an active protein. More particularly, the present invention is concerned with the production of an active protein by linking two peptide fragments, at least one of said two peptide fragments being one which has been obtained by recombinant DNA technique or has been obtained by a method comprising producing a predetermined peptide fragment by recombinant DNA technique and deleting from or adding to said predetermined peptide fragment at its N-terminus at least one amino acid residue. By this method, there can be easily, safely obtained a desired active protein in a skillful manner.
The term "active protein" used herein means a protein of a three dimentional structure which comprises 60 or more of amino acid residues and exhibits a physiological activity.
In recent years, there have been established the structures of various physiologically active peptides, and studies have been made in the art to synthesize such active peptides. Such peptides may be classified, according to the number of amino acid residues constituting the peptide, into oligopeptides (2 to about 10 amino acid residues), polypeptides (about 10 to about 60 amino acid residues) and proteins (about 60 or more amino acid residues) and, therefore, oligopeptides, polypeptides and proteins used in the present specification mean such peptides as classified above.
In 1881, the synthesis of a dipeptide, the smallest peptide, was reported [T. Curtius, J. Pract. Chem., 24, 239 (1881)]. Since then, various attempts to produce peptides were made. In 1969, bovine RNase A was organochemically synthesized by a solid phase method [B. Gutte and R. B. Merrifield, Journal of American Chemical Society, 91, 501 (1969)] but the end product was not obtained in pure form. On the other hand, H. Yajima and N. Fujii succeeded in organic synthesis of bovine RNase A by a liquid phase method after the study of a period of time as long as three and a half years [H. Yajima and N. Fujii, Chemical and Pharmaceutical Bulletin, 29, 600 (1981)].
In spite of the success of Yajima et al., techniques have not yet progressed to an extent that proteins are easily synthesized. In general, the technique of organic synthesis for a protein is on the extension of the method of E. Fisher, Berichte Deutsche Chemische Gesellschaft, 40, 1754 (1907). According to the general technique, however, extremely increasing difficulties will be encountered in the synthesis of a protein with the increase of size of the protein. Polypeptides differ from proteins mainly in size and properties. Although the synthesis of proteins is an extension of the synthesis of polypeptides, difficulties not accompanying the synthesis of polypeptides, for example, the synthesis of human insulin P. Sieber et al., Helv. Chim. Acta., 57, 2617 (1974)] are encountered in the synthesis of proteins. In the liquid phase method, a long chain peptide of which the functional groups are entirely or partially protected usually becomes sparingly soluble with the extension of peptide chain, so that a large amount of solvent is required for the synthesis reaction of a protein. However, the use of a large amount of solvent causes the reactivity of the reactants in the intended reaction to be lowered. When the reaction is carried out under drastic conditions in order to avoid lowering of the reactivity, racemization and/or other side reactions tend to occur to a large extent so that it becomes difficult to obtain the intended product. On the other hand, in the solid phase method, the above problems are not involved. However, in the solid phase method, proteins are synthesized through many steps of reactions so that defective peptides formed due to incomplete reactions accumulate. In such case, it is difficult to remove the accumulated defective peptides at the final purification step. In either method, a long time is required to synthesize proteins because the synthesis of protein is performed by bonding necessary amino acids one by one.
As is apparent from the above, it is difficult to synthesize proteins completely artificially. So, as a more convenient method of the synthesis of proteins, there was proposed a semisynthetic method in which a peptide fragment of the kind which can be easily synthesized is first synthesized, and then mixed with or linked to a natural protein fragment extracted, through the partial decomposition of the natural protein, from the living body to obtain a protein having a physiological activity [K. Hofmann, Journal of American Chemical Society, 88, 4107 (1966)] [(A. Komoriya et al., International Journal of Peptide and Protein Research, 166, 433 (1980)]. However, this method does not have any industrial value, because the proteins obtained in this method are not those which have been newly provided but those which have been obtained by partially decomposing natural proteins, followed by reconstruction through semisynthesis. As another semisynthetic method, although it is restricted to a special case, there was proposed a method in which a synthetic octapeptide is bonded to swine insulin fragments by means of an enzyme to obtain human insulin [K. Inouye et al., Journal of the American Chemical Society, 101, 751 (1979)]. This enzymatic method which is useful in synthesis of human insulin was reported in M. Bergmann and H. Francel Conrat, Journal of Biological Chemistry, vol. 124, p. 1 (1983). Recently, the usefulness of the enzymatic method was re-confirmed. In this respect, reference may be made to Japanese Patent Application Laid-Open Specification No. 51-110094/1976; Japanese Patent Application Laid-Open Specification No. 53-62896/1978; Y. Isowa et al, Bulletin of Chemical Society of Japan, vol. 50, pp. 2762 and 2766 (1977); K. Morihara and T. Oka, Biochemical Journal. vol. 162, p. 531 (1977); R. W. Sealock and M. Laskowski, Biochemistry, vol. 8, p. 3703 (1969); and G. A. Homandberg et al, ibid., vol. 17, p. 5220 (1978). It is to be noted, however, that while the enzymatic method is useful where the difference between the desired peptide of an organism and an available corresponding peptide of another organism resides in the kind of a single amino acid residue only, the method is not generally applicable to the synthesis of a protein since differences with respect to a plurality of amino acid residues exist between the desired protein and an available protein. Moreover, apart from the applicability of the enzymatic method, the use of natural protein fragments obtained by extraction as the raw material is disadvantageous from the viewpoints of availability, purity, danger of contamination with viruses and the like.
According to the progress of recombinant DNA technique, the syntheses of various active peptides using synthetic or natural DNA have been reported following the pioneering work by Goeddel and Itakura [see D. V. Goeddel and K. Itakura: Proceeding National Academy of Science in U.S.A., Vol. 76, p. 106 (1979)]. The syntheses of active peptides by recombinant DNA technique, however, are accompanied by the following drawbacks. First, a large amount of microorganisms or cells which serve to produce active peptides are propagated during the production of active peptides, and it is necessary to dispose of the propagated microorganisms or cells. For the purpose of safety, it is requisite that before the disposal, all of such propagated microorganisms or cells be killed. However, it is very difficult to accomplish this on a commercial scale. Secondly, since the initiation codon for peptide synthesis is identical with the codon of a methionine residue, a peptide directly synthesized by recombinant DNA technique inevitably has a methionine residue at the N-terminus thereof even if the corresponding natural peptide which is desired to be obtained does not have a methionine residue at the N-terminus thereof. Hence, a synthetic peptide obtained by this method cannot be identical with the desired natural peptide with respect to the amino acid residue at the N-terminus.
As an improved method for the synthesis of a physiologically active peptide by recombinant DNA technique, there has heretobefore been proposed a method in which a precursor having the cleaving site at a position of an arginine residue, lysine residue or methionine residue is synthesized by recombinant DNA technique, and the so-synthesized precursor is cleaved by treating it with trypsin, chymotrypsin or cyanogen bromide in accordance with the known method described in G. R. Stark et al., Journal of Biological Chemistry, vol. 235, p. 3177 (1960), and in E. Gross and B. Witkop, Journal of Biological Chemistry, vol. 237, p. 1856 (1962), thereby to obtain a physiologically active peptide (Japanese Patent Application Laid-Open Specification No. 54-145289/1979). The above-mentioned improved method, however, has the following two defects. One of such defects is that in the above-mentioned method, peptide fragments produced by cleavage of the precursor include not only the intended peptides but also undesirable peptide fragments, because the precursor comprises the intended peptide and a peptide fragment attached to the N-terminus of the intended peptide. Another defect is that application of the above-mentioned method is restricted only to the synthesis of an active peptide which does not contain an arginine residue, lysine residue or methionine residue, because if the intended peptide contains an arginine residue, lysine residue or methionine residue, the intended peptide is cut into pieces simultaneously with the desired cleavage. Therefore, this method cannot apply to the synthesis of peptides comprised of a large number of amino acids, such as proteins, since such peptides generally contain an arginine residue, lysine residue or methionine residue.
As another method in which artificial cleavage is involved, there is known a method in which a precursor having an innate cleaving site is produced in a cultured cell of a higher animal, and the so-produced precursor is caused to be cleaved in said cell at the innate cleaving site thereof, thereby to obtain an active peptide [P. W. Gray et al, Nature, vol. 195, p. 503 (1982); R. Devos et al, Nucleic Acid Research, vol. 10, p. 2487 (1982); Japanese Patent Application Laid-Open Specification No. 58-90514/1983; and T. Taniguchi et al, Nature, vol. 302, p. 305 (1983)]. According to this method, an active protein not having methionine at the N-terminus thereof can be synthesized, but the productivity is extremely low and, hence, this method cannot apply to the industrial-scale synthesis of an active peptide.
As an improvement of the above-mentioned method in which a precursor is produced in a cultured cell, there is known a method in which a precursor having an innate cleaving site is produced using, in place of a cultured cell of a higher animal, a yeast which is a eucaryotic cell of a microorganism, and the so-produced precursor is cleaved in said yeast at the innate cleaving site thereof, thereby to obtain an active peptide [R. A. Hitzeman et al, Science, vol. 219, p. 520 (1983)]. However, in this method, the yeast serves to cleave the precursor at the innate cleaving site thereof, but it also cleaves the precursor at a site other than the innate cleaving site and, hence, if the synthesis of an active peptide is effected by using this method, there is produced a mixture of peptides which are different in length. In the case of the above-mentioned method in which a precursor is cleaved in the yeast, there is such a disadvantage that active peptide-producing yeasts are caused to be produced in large quantities. Therefore, when the synthesis of an active peptide is effected by this method, it is requisite that, for the purpose of safety, all of such peptide-producing yeasts be killed.
On the other hand, as a method for removing only the methionine residue attached to the N-terminus of the intended peptide produced by recombinant DNA technique while leaving other methionine residues, if any, in the intended peptide as they are, the present inventor established a method of removing only the methionine residue attached to the N-terminus of the peptide by the so-called phenylisothiocyanate method or the so-called aminopeptidase method (Japanese Patent Application Laid-Open Specification No. 58-110548/1983). The phenylisothiocyanate method is disclosed in P. Edman, Acta Chemica Scandinavia, 4, 227, (1950) and the aminopeptidase method is disclosed in D. H. Spackman et al, Journal of Biological Chemistry, 212, 255 (1955) and E. D. Wacksmuth, Biochemistry, 5, 169 and 175 (1966). However, this method has a disadvantage that not only the intended peptide is denatured but also other amino acid residues in the amino acid sequence of the peptide as well as the methionine residue attached to the N-terminus of the intended peptide are removed successively from the N-terminus of the intended peptide.
In summary, conventionally, when a protein composed of amino acid residues as many as about 50 or more is intended to be produced by organic synthesis, there is a disadvantage in that many complicated reaction steps are needed and a long period of time is required to produce peptides. Further, when the amino acid residues to be linked have low reactivity, even if they are reacted the reaction yield is low and it is necessary to isolate the desired product from the raw materials remaining unreacted in the reaction step. Moreover, as mentioned above, when a protein composed of amino acid residues as many as about 50 or more is intended, the synthesis method inevitably involves many complicated steps of reactions, leading to occurrence of side reactions in each step. As a result, the yield of the intended product is lowered and it is necessary to isolate the intended product from a large amount of the by-products. The isolation is very difficult to perform.
On the other hand, when a protein having a large molecular weight is intended to be produced directly by recominant DNA technique with respect to the entire amino acid sequence of the protein, the peptide which contains at least one methionine residue in its amino acid sequence but does not have a methionine residue as the N-terminal amino acid residue cannot be produced. This is because a product prepared by recombinant DNA technique inevitably has a methionine residue as the N-terminal amino acid residue for the reason as set forth before and, as mentioned above, the selective removal of only such a methionine residue attached to the N-terminus of the intended protein is extremely difficult. Further, it is dangerous if the microorganisms or cells used in the culturing step for producing the intended protein are released out of the culture system and, therefore, it is necessary to kill all of the used microorganisms or cells in the culture system after culturing. However, it is difficult to kill all of the microorganisms or cells on an industrial scale.
The present inventor has made extensive and intensive studies in order to overcome the above-mentioned defects of the conventional methods. As a result, the present inventor has found that the intended active protein can be easily and safely produced by providing two peptide fragments, at least one of which is one which has been obtained by recombinant DNA technique or has been obtained by a method comprising preparing a predetermined peptide fragment by recombinant DNA technique and deleting from or adding to said predetermind peptide fragment at its N-terminus at least one amino acid residue, and linking said two peptide fragments to each other. The present invention has been made based on such a novel finding.
Therefore, it is a primary object of the present invention to provide a novel method for easily and safely producing an active protein on an industrial scale.